RNA干扰抑制环氧化酶-2表达对宫颈癌HeLa细胞增殖的影响

Effect of COX-2 Expression Inhibition by RNA Interference on Proliferation of Cervical Carcinoma Cell Line HeLa

目的建立以小分子干扰RNA(siRNA)抑制环氧化酶-2(COX-2)表达的宫颈癌HeLa细胞模型,探讨COX-2在肿瘤细胞增殖、凋亡方面的作用和可能机制。方法构建靶向抑制COX-2的siRNA表达载体,转染HeLa细胞,Western blot法确定筛选的稳定表达siRNA的转化克隆COX-2表达受抑制;流式细胞仪检测RNA干扰(RNAi)抑制COX-2表达后细胞凋亡和细胞周期的变化;克隆形成试验检测细胞克隆形成率;RT-PCR及Western blot法检测ku70、ku80的表达变化。结果获得稳定表达siRNA的转化细胞株(HeLa/COX-2i),其COX-2蛋白表达明显受抑制,而转入阴性对照质粒的细胞株(HeLa/Negi)COX-2蛋白表达不受影响;HeLa/COX-2i细胞的克隆形成率[(45±4)%]显著低于HeLa细胞[(85±7)%](P<0.01);HeLa,HeLa/Negi,HeLa/COX-2i 3株细胞的细胞周期分布以及细胞凋亡率差异无显著性意义;RT-PCR及Western blot显示RNAi抑制COX-2表达后ku70、ku80在转录和翻译水平均受抑制。结论RNAi抑制COX-2表达后可抑制HeLa细胞增殖,其机制可能与ku70、ku80的表达下调有关。

Objective To inhibit cyclooxygenase-2 （COX-2） expression in cervical carcinoma cell line HeLa with small interfering RNA （siRNA）, and explore its role and mechanism on cell proliferation and apoptosis. Methods Plasmid targeting COX-2 was constructed and transfected into HeLa cells. COX2 protein level was examined by Western blot. Flow cytometry （FCM） was performed to analyze the change of the apoptosis and cell cycle distribution. The proliferation of HeLa cells was determined by clone formation assay. RT-PCR and Western blot were used to detect the expression of ku70 and ku80. Results （1） The COX-2 protein expression in stably transfected cell line -- HeLa/COX-2i was suppressed markedly, but not changed in control cells -- HeLa/Negi. （2） The clone formation efficiency was significantly lower in HeLa/COX-2i cells [（45±4）%1 than in control cells [ （85± 7）%] （P〈0.01）. （3） The cell cycle distribution and the apoptosis rate had no change after transfection of plasmid targeting COX-2. （4） The mRNA and protein levels of ku70 and ku80 were significantly decreased after transfection of plasmid targeting COX-2. Conclusion COX-2 can inhibit the proliferation of HeLa cells possibly by suppressing the ku70 and ku80 expression.